We have continued our analysis of the mechanism(s) by which a single amino acid substitution in 15-25% of CHO cells' glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polypeptides results in altered morphology, mobility and distribution of late endocytic organelles. Our results indicate that both mutant and wild type GAPDH are bound to microtubules in semi-intact cell preparations. In cells expressing mutant GAPDH, organization of the cytoskeleton is altered: microtubule bundles are present, thin filaments, as yet unidentified, extend from these bundles and appear to attach to endosomal compartments clustered at the cell poles. The mutant GAPDH we are studying contains a substitution at an amino acid that has been conserved throughout evolution. We have examined the effects of this mutation on enzyme function, comparing purified homotetramers of mutant and wild type GAPDH. The mutant enzyme manifests one-half the normal activity; no significant differences were observed in either substrate or cofactor concentration dependence. A hallmark of the mutant enzyme is that its binding to microtubules is resistant to dissociation by ATP; in contrast, its enzymatic activity shows a >50-fold increase in sensitivity to ATP.